The prion protein (PrP), a brain glycoprotein involved in various neurodegenerative diseases, has proven to be a particularly instructive example of complex and highly regulated translocation. In addition to its notoriety as the putative "protein-only" infectious agent in prion diseases, the biogenesis of PrP at the ER is unusual in that an initially homogeneous cohort of nascent PrP chains gives rise to four distinct topologic forms: a fully translocated form (termed secPrP), two transmembrane forms that span the membrane in opposite orientations (NtmPrP and CtmPrP), and a cytosolic form (cyPrP). In vivo studies have revealed that even a slight overrepresentation of the CtmPrP topologic form results in the development of neurodegenerative disease in both mouse model systems and naturally occurring human disease. Furthermore, cyPrP can be both aggregation-prone and neurotoxic under some circumstances. [unreadable] [unreadable] To gain insight into how these variants are initially generated, we are dissecting the pathways of PrP biogenesis and degradation. Our results indicate that the decisive event in avoiding the generation of both of the potentially harmful forms of PrP (CtmPrP and cyPrP) is the signal sequence-mediated translocation of the N-terminus of PrP into the ER lumen. By using a constitutive, highly efficient signal sequence, the generation of CtmPrP and cyPrP can be substantially reduced. The consequences of this manipulation in a cultured neuronal cell line are a marked reduction of cytotoxic and aggregation-prone variants of PrP and protection from apoptotic cell death. These results define a pathway for the normal biogenesis of PrP, and demonstrate that the total cellular burden of cytotoxic forms of PrP is controlled primarily during its initial translocation into the ER. Transgenic mice have now been created to determine whether CtmPrP-mediated neurodegeneration and cyPrP-mediated neurodegeneration can be averted in vivo by modulating this newly discovered step during PrP biogenesis. We are also investigating the pathways by which the various forms of PrP are normally metabolized by the cell to determine whether modulation of these events are involved in the progression of neurodegeneration. It is anticipated that a combination of defects in biosynthesis and/or clearance of certain forms of PrP collaborate to eventually cause neuronal dysfunction and death. Conversely, manipulation of these events may be able to slow or reverse the neurodegenerative process in these diseases.[unreadable] [unreadable] Parallel biochemical studies employing the solubilization, fractionation and reconstitution of ER membrane proteins have demonstrated that regulatory trans-acting factors are absolutely required for PrP to be synthesized in the proper ratio of its topologic forms. We have now purified two of these factors and identified them as the translocon-associated protein complex (TRAP) and protein disulfide isomerase (PDI). Analysis of PrP translocation intermediates suggests that TRAP and PDI act sequentially to facilitate translocation of PrP's N-terminus into the ER lumen, the decisive event in determining its topology. Ongoing studies are investigating the role of these newly discovered factors in the biogenesis of other substrates and their potential role in the pathogenesis of PrP-associated neurodegeneration.